Evaluation of a large-scale aptamer proteomics platform among patients with kidney failure on dialysis

Background Patients with kidney failure suffer high mortality, and we currently lack markers for risk stratification for these patients. We carried out a quality control study of a modified aptamer assay (SomaScan v.4.0) that measures ~ 5000 proteins, in preparation for a larger study using this platform in cohorts with kidney failure. Methods Forty participants from the Cardiac, Endothelial Function and Arterial Stiffness in End-Stage Renal Disease (CERES study) were selected to analyze technical and short-term biological variability, orthogonal correlations and differential protein expression in plasma from patients who died during 2.5 year follow-up. Long-term (one year) variability was studied in 421 participants in the Chronic Renal Insufficiency Cohort. We evaluated 4849 aptamers (4607 unique proteins) using data formats including raw data and data formatted using Adaptive Normalization by Maximum Likelihood (ANML), an algorithm developed for SomaScan data in individuals with normal kidney function. Results In ANML format, median[IQR] intra-assay coefficient of variation (CV) was 2.38%[1.76, 3.40] and inter-assay CV was 7.38%[4.61, 13.12]. Short-term within-subject CV was 5.76% [3.35, 9.72]; long-term CV was 8.71%[5.91, 13.37]. Spearman correlations between aptamer and traditional assays for PTH, NT-proBNP, FGF-23 and CRP were all > 0.7. Fold-change (FC) in protein levels among non-survivors, significant after Bonferroni correction, included SVEP1 (FC[95% CI] 2.14 [1.62, 2.82]), keratocan (1.74 [1.40, 2.15]) and LanC-like protein 1 (0.56 [0.45, 0.70]). Compared to raw aptamer data, technical and short-term biological variability in paired samples was lower in ANML-formatted data. ANML formatting had minimal impact on orthogonal correlations with traditional assays or the associations of proteins with the phenotype of mortality. Conclusions SomaScan had excellent technical variability and low within-subject short-term variability. ANML formatting could facilitate comparison of biomarker results with other studies that utilize this format. We expect SomaScan to provide novel and reproducible information in patients with kidney failure on dialysis.

Previous versions of the SomaScan Assay have been applied successfully to biomarker discovery and validation in many pharmaceutical research and development projects, diagnostics discovery and development projects and academic research projects.A selected list of peer-reviewed articles can be found here.

SOMAmer reagents are discovered using robust SELEX technology
SOMAmer reagents are single stranded DNA-based protein affinity reagents that benefit from aptamer technology developed over the past 30 years 1,2 .The more recent proprietary innovation incorporates chemically modified nucleotides (Figure 1) that facilitate the binding to proteins, expanding the chemical diversity of standard aptamers and enhancing the specificity and affinity of protein-nucleic acid interactions 3 .These modified nucleotides are incorporated into nucleic acid libraries used for the iterative selection and amplification process called SELEX (Systematic Evolution of Ligands by EXponential enrichment) from which SOMAmer reagents are selected [4][5][6] .Using a novel, proprietary SELEX process, SomaLogic has generated SOMAmer reagents to proteins that had been resistant to selection with unmodified nucleic acids (ACTG traditional aptamers) 3 .These chemical modifications in turn allow the SOMAmer reagent to tightly bind its target protein in ways never before possible 7 .A key advantage of this artificial selection process is that conditions can be tailored to select for desirable properties, including specificity, slow off-rate, and specific assay conditions.SOMAmer reagents are selected against proteins in their native folded conformations and are therefore generally found to require an intact, tertiary protein structure for binding.
As such, unfolded and denatured-and therefore presumably inactive-proteins are not detected by SOMAmer reagents.Crystal structures of SOMAmer reagents bound to their cognate protein targets indicate that the modified nucleotides contribute extensively to intramolecular contacts within the SOMAmer reagent and to intermolecular contacts with the protein targets 6 .
In Figure 2, the X-ray crystal structure of the PDGF-BB SOMAmer reagent bound to PDGF-BB demonstrates that the interactions between the SOMAmer reagent and its cognate protein are mainly mediated via the modified nucleotides.The co-crystal structures show very specific interactions between the SOMAmer reagent and target with binding site dimensions of 1100-1200 Å 2 , similar to antibody-antigen interactions 8 .
The dissociation kinetics of a subset of SOMAmer reagents binding to their respective targets were determined using a solution-phase radiolabeled binding assay 3 , and a subset were confirmed using the Biacore Flexchip surface plasmon resonance biosensor.Biosensor results confirm slow dissociation off-rates, in the 10 -5 to 10 -4 s -1 range, that correlate well with dissociation rate constants measured by solution-phase radiolabeled binding assays.Overall, SOMAmer reagents are analogous to high-quality antibodies that recognize intact tertiary protein structures.
However, since they are made out of nucleic acids, SOMAmer reagents have several advantages over antibodies, such as tailored in vitro selection conditions, chemical synthesis, storage stability, and detection using sensitive and advanced DNA detection methods.

SOMAmer Reagents to Approximately 5,000 Human Proteins
SOMAmer reagents have been created for human protein targets that cover a diverse set of biological processes, including cancer, inflammation and cardiovascular function, to name a few.Targets to date extensively cover major molecular functions including receptors, kinases, growth factors and hormones, and span a diverse collection of secreted, intracellular and extracellular proteins or domains.

SOMAmer Reagents are Chemically Synthesized, Stable and Rigorously Analyzed
After identification using SELEX technology, the SOMAmer reagents are chemically synthesized, purified and analyzed by methods including ultra high-performance liquid chromatography (UPLC), capillary gel electrophoresis (CGE) and mass spectrometry (MS).Extensive functional analysis ensures consistent high performance of the SOMAmer reagents as quantitative affinity reagents.In order to test the specificity of SOMAmer reagents used in the SomaScan Assay for their respective initial target proteins, we perform a variety of characterization steps.

These steps include:
• In silico selection, procurement (when available), and direct SOMAmer reagent binding experiments in buffer with related proteins.
• Pull-down assays followed by MS-based and SDS gel-based analyses of the protein(s) bound by the SOMAmer reagent from biological matrices.
All types of affinity reagents (antibodies, traditional aptamers, etc.) are subject to specificity issues.Recognizing how critical the accuracy of the SomaScan Assay is for both research and clinical purposes, SomaLogic is committed to regular assessment of SOMAmer reagent specificity, and to transparency in communicating the results of those ongoing efforts.
A SOMAmer-protein binding step is followed by a series of partitioning and wash steps to convert relative epitope concentrations into measurable nucleic acid signals that are quantified using DNA-hybridization microarrays.Assay details are provided in the Appendix and in Gold et al., 2010 3 .The readout in relative fluorescent units (RFU) is directly proportional to the amount of target epitope in the initial sample.
Achieving the 10 10 Dynamic Range: SOMAmer Reagent Mixes The large dynamic range of the SomaScan Assay in plasma and serum results from the optimization of SOMAmer reagent measurements across three serial dilutions of the sample (Figure 4).A specific SOMAmer reagent is only present in one of the three dilution groups.The least concentrated sample is designed to detect the most abundant proteins, and the most concentrated solution is designed to detect the least abundant proteins.
Based on reported literature values, the SomaScan Assay robustly measures analytes spanning a range of 10 logs in human plasma or serum (e.g.albumin to interleukins or interferons).

SomaScan Assay Characterization
The SomaScan Assay v4.0 has been validated in human EDTA plasma, serum, and urine.
The SomaScan Assay has excellent reproducibility.In plasma, half of the SOMAmer reagents demonstrated a median Coefficient of Variation (CV) less than 5.0% and only 10% of the SOMAmer reagents demonstrated a median CV of 11.8% or higher.All values were determined in the multiplex assay, profiling approximately 5,000 reagents simultaneously.SomaScan Assay metrics using urine can be found in our Urine Measurements on the SomaScan Platform Technical Note (SL00000472).
By measuring thousands of proteins at a time in each blood sample with the SomaScan Platform, it is possible to uncover a precise set of specific protein changes that provide information on current status and future trajectory for virtually every disease or condition of interest.For example, we can find repeatable patterns of protein changes that are associated with clinical indications (e.g., cardiovascular events) or fitness attributes (e.g., VO 2 Max).
Each set of specific protein patterns, in turn, becomes the basis of a specific SomaSignal Test that can be ordered by researchers who desire clinical assessments of study participants.The current suite of available RUO SomaSignal Tests can be found on the SomaLogic website.

SomaLogic Quality Systems
The SomaScan Assay is performed under the SomaLogic Quality System (QS) in a laboratory that follows CLIA standards for a laboratory developed test.
The assay is performed in a facility that contains both access and environmental control.Equipment within the facility is maintained, calibrated, and operated in compliance with controlling Standard Operating Procedures (SOPs).Equipment and associated software are validated for their intended uses in support of the SomaScan Assay.
Method validation has been completed for processes that could impact the performance of the SomaScan Assay.SOPs cover the incoming receipt, inspection, and release of raw materials to ensure that the materials used in the production of assay reagents or directly in the assay maintain the performance requirements established during the development of the SomaScan Assay.
Step 2 SOMAmer reagents bound to streptavidin beads are used to capture proteins from a complex mixture of proteins (tan).Step 6 A polyanionic competitor (green) prevents rebinding of non-specific complexes.
Step 8 SOMAmer reagents are released from the complexes by denaturing the proteins.

Summary
The SomaScan Platform is a powerful, highly multiplexed platform for discovering novel biomarkers during drug discovery, pre-clinical and clinical drug development, and for the development of clinical diagnostics, across a wide range of clinically important diseases.
Table 1 summarizes the SomaScan Assay v4.0 metrics in plasma.SomaLogic's SomaScan Platform technology provides significant advantages in sample size, cost, time, multiplexing capability, measurement range, and flexibility of readout over many alternate protein biomarker platforms.Plate scaling adjusts for total signal variation that occurs from plate to plate, typically associated with scanner intensity differences.The median value of the calibrator reference ratios is used to calculate a single scale factor for an entire plate.population standard deviations of the normal reference; the process is iterated to convergence and maximizes the probability that a sample's RFU measurements come from the reference distribution.The scale factor for each dilution bin is then applied to their respective SOMAmer reagents.This step is applied to QC and individual samples.

Sensitivity in
Note: Normalization to a reference standardizes the overall signal from every sample and is appropriate for experiments where all samples should have approximately the same amount of total protein.
For samples with inherently different protein amounts (e.g.Duchenne Muscular Dystrophy or leukemic blood), median signal normalization to a reference will mute variations in protein levels that may be of interest.For further details, please refer to the SomaLogic Technical Note SL00000063.

Figure 2 .
Figure 2. X-ray crystal structure of a SOMAmer reagent bound to PDGF-BB.Modified nucleotides are shown in purple, and the DNA backbone and unmodified bases are in green.

Figure 4 .
Figure 4. SomaScan Assay dynamic range.SOMAmer reagent mixes are prepared to achieve optimal detection in mixtures with a large range of concentrations.Shown here is the dilution distribution of SOMAmer reagents for SomaScan Assay v4.0 with plasma and serum.
Calibration reduces the variability between runs and/or entire experiments on a SOMAmer-by-SOMAmer reagent basis.The ratio of the SOMAmer-specific reference value to the median value of the Calibrator Controls is the calibration scale factor for the SOMAmer reagent across the run.Adaptive normalization to a reference, Adaptive Normalization by Maximum Likelihood (ANML), adjusts for inter-sample technical and biological variability in total signal within and between runs.A scale factor for each set of SOMAmer reagents within a dilution bin is computed using only sample values within two SomaLogic®, SOMAmer®, SomaScan® and associated logo are registered trademarks of SomaLogic, Inc. © 2020 SomaLogic, Inc. |2945 Wilderness Pl.Boulder, CO 80301 | www.somalogic.com| SL00000442R2 Republished from Somalogic Technical Note under a CC BY license, with permission from Somalogic, original copyright 2021

Table 1 .
Summary of SomaScan v4.0 Assay metrics to human targets.Quality control check uses replicate Quality Controls (QC) that are pooled matrix-matched samples (e.g.plasma, serum) run in the SomaScan Assay alongside clinical samples to quantify the quality of each assay run by determining the accuracy of the median replicate signal for each SOMAmer reagent compared to the reference.QC check is performed after steps 1-5 above have been applied.Hybridization normalization mitigates bias caused by differential readout conditions (eluate transfer, hybridization, wash, scan) in individual microarrays.Control SOMAmer reagents are not exposed to protein but added during the transfer of SOMAmer reagents to the Agilent slide.The control signals are then used to calculate a single scaling factor that is applied to all spots in the microarray.Intraplate median signal normalization was developed to account for differences in total signal between replicate control samples.It uses measurements from Calibrator Controls to calculate a sample-based scaling factor for each of the three dilution groups.
SL: 00000046 Rev 2: 2021-01 SomaScan Assay v4.0 Technical Note 6.Like the Quality Controls, the Calibrator Controls are matrix-matched samples (e.g.plasma, serum) that are run in replicate in the SomaScan Assay alongside clinical samples.Plate scaling and calibration adjustments require data from invariant controls that are run in replicate in all assays.These data are used in conjunction with references generated during assay qualification to calculate a scaling factor for every plate and SOMAmer reagent.